Method and test kit for the detection of insecticide resistance in insect vectors

ABSTRACT

A method for checking for elevated activity of mixed-function oxidases using a single insect, which comprises homogenizing a single insect in an acid pH buffer, preferably by grinding it in a small tube, such as an Eppendorf tube, with a pestle, adding a buffered tetramethylbenzidine solution as substrate, adding hydrogen peroxide as oxidant, and checking the blue coloration, the intensity of which indicates the degree of elevated mixed function oxidase activity. The invention also provides a test kit for use in the method which comprises separate containers of:  
     I. an acid pH buffer, preferably pH4 buffer  
     II. tetramethylbenzidine  
     III. a solvent for tetramethylbenzidine, preferably methanol, and  
     IV. hydrogen peroxide.

[0001] This invention relates to a method and test kit for determininginsect resistance to insecticides and more particularly provides amethod and test kit for field use for determining insect resistance topyrethroid based insecticides.

BACKGROUND TO THE INVENTION

[0002] Chemical insecticides remain as the most important agents for thecontrol of vector-borne diseases such as malaria, dengue, filariasisetc. Countless deaths from these diseases have been spared since theintroduction of the first synthetic insecticide, DDT in the 1940's.Prolonged use of insecticides has however, induced the development ofresistance in the vectors, primarily insects, that carry such diseases,which renders them less effective in combating the vectors and thus thediseases that they spread. Resistance detection is mainly based on theWorld Health Organisation (WHO) standard test procedures which, amongother things, are not user-friendly, use test kits of short shelf lifeand need skilled manpower to conduct the tests and interpret the testresults. As a result, very often little is known of the susceptibilitystatus of many insect vectors, which tends to affect the outcomes ofcontrol programmes.

[0003] The development of rapid field test kits would be a majorbreakthrough in the control of vector-borne diseases.

[0004] In general, the biochemical basis of resistance is due to 3mechanisms:

[0005] 1) increased level of non-specific esterases targeted againstorganophosphates and carbamates;

[0006] 2) elevated activity of mixed-function oxidases againstpyrethroids; and

[0007] 3) non-susceptability of acetylcholine esterase againstorganophosphates and carbamates.

SUMMARY OF THE INVENTION

[0008] This invention provides a method and test kit for determiningelevated activity of mixed-function oxidases in insects using a singleinsect that is quick and easy to use and produces results that are verysimple to interpret.

[0009] According to the invention the test kit comprises a containercontaining separate containers of:

[0010]1. an acid pH buffer

[0011] II. tetramethylbenzidine

[0012] III. a solvent capable of dissolving tetramethylbenzidine and

[0013] IV. hydrogen peroxide

[0014] For field use the kit will also contain the equipment forcarrying out the test, for example mixing containers, test tubes, forexample Eppendorf tubes, a pipette and a micro-assay plate and, usually,a colour chart.

[0015] The buffered pH is preferably 4 to 6, more preferably about 5.

[0016] The pH buffer is preferably sodium acetate.

[0017] The solvent is preferably absolute methanol.

[0018] The hydrogen peroxide is preferably used as a 3% w/w aqueoussolution.

[0019] The method of the invention comprises homogenizing a singleinsect in the buffer, preferably by grinding it in a small tube, such asan Eppendorf tube, with a pestle, adding buffered tetramethylbenzidinesolution as substrate, adding hydrogen peroxide as oxidant, and checkingthe blue coloration, the intensity of which indicates the degreeelevated mixed function oxidase activity, and hence resistance topyrethroid based insecticides.

[0020] The coloration for a non-resistant insect is at most very faintand increases to dark blue as the degree of resistance increases.

[0021] The degree of coloration can be estimated by eye, by comparisonwith a colour chart or, if more accurate results are needed, by scanningwith an immuno-assay reader at 630 nm.

[0022] The invention has the advantage of being the first test kitavailable for testing for elevated activity of mixed-function oxidasesthat requires only a single insect for testing and that is used in amethod that gives rapid test results that are obtainable virtuallyinstantaneously and which can be read visually and calorimetricallywithout equipment. The test kits and method require less skilledmanpower and are easy to use without special training. Moreover, thekits have a long shelf life and are stable for weeks at room temperature

[0023] Use of the kit will:

[0024] 1. considerably simplify resistance detection

[0025] 2. ensure that chemical insecticides used to control pests anddisease vectors are effective and hence save lives, cost and manpower

[0026] 3. allow constant and regular monitoring of the susceptibility oftarget insects and

[0027] 4. assist in the design of new control agents, counter-measuresand understanding of the mode of action of insecticides.

DESCRIPTION OF PREFERRED EMBODIMENT

[0028] Five bottles were prepared containing, respectively 10 ml ofsodium acetate buffer (pH 5), 2.5 ml of absolute methanol, 5 mgtetramethylbenzidine, 7.5 ml of sodium acetate buffer (pH 5) and 5 mlhydrogen peroxide (3%)

[0029] To carry out the test, the absolute methanol is poured into thetetramethylbenzidine and shaken to dissolve the tetramethylbenzidinecompletely and the resulting solution is buffered with the 7.5 ml ofsodium acetate buffer. A single mosquito is homogenized in one drop ofthe buffer in an Eppendorf tube using a pestle and further diluted withbuffer to the 1 ml mark on the Eppendorf tube. Two drops of thehomogenisate are dropped into each well of a micro-assay plate followedby four drops of the tetamethylbenzidine solution and one drop of thehydrogen peroxide.

[0030] The results were assessed by eye scoring, by colour checkingagainst a calibrated colour chart or by scanning using an immunoassayreader at 630 nm.

1. A method for checking for elevated activity of mixed-functionoxidases in insects, which comprises homogenizing a single insect in anacid pH buffer, adding acid pH buffered tetramethylbenzidine solution assubstrate, adding hydrogen peroxide as oxidant, and checking the bluecoloration, the intensity of which indicates the degree of elevatedmixed function oxidase activity.
 2. A method according to claim 1carried out using a test kit comprising separate containers of: I. anacid pH buffer II. tetramethylbenzidine III. a solvent fortetramethylbenzidine and IV. hydrogen peroxide.
 3. A method according toclaim 1 or claim 2, wherein the buffer is a pH 4 to 6 buffer.
 4. Amethod according to claim 3, wherein the buffer is a pH 5 buffer.
 5. Amethod according to claim 1 or claim 2, wherein the buffer is sodiumacetate.
 6. A method according to any one of claims 1 to 5, wherein thesolvent is acetone.
 7. A method according to any one of claims 1 to 6,wherein the hydrogen peroxide is a 3% w/w aqueous solution.
 8. A testkit for use in the method according to any one of claims 1 to 7comprising separate containers of: I. an acid pH buffer II.tetramethylbenzidine III. a solvent for tetramethylbenzidine and V.hydrogen peroxide.
 9. A test kit according to claim 8, wherein thebuffer is a pH 4 to 6 buffer.
 10. A test kit according to claim 9,wherein the buffer is a pH 5 buffer.
 11. A test kit according to claim8, wherein the buffer is sodium acetate.
 12. A test kit according to anyone of claims 8 to 11, wherein the solvent is methanol.
 13. A test kitaccording to any one of claims 8 to 12, wherein the hydrogen peroxide isa 3% wlw aqueous solution.
 14. A test kit according to any one of claims8 to 13, which also contains equipment for carrying out the test.
 15. Atest kit according to claim 14, which contains test tubes, a pipette anda micro-assay plate.
 16. A test kit according to claim 14, which alsocontains a colour chart.